The long term objective of this research is to understand the basic biochemical mechanisms that underlie pre-mRNA splicing. The removal of introns from pre-mRNA is required for the expression of most genes in eukaryotes. The expression of some genes is regulated by splicing, and the generation of protein isoforms frequently occurs through alternative splicing. Many mutations that lead to genetic diseases in humans affect pre-mRNA splicing. Therefore, unraveling the mechanism of splicing is of great importance for human health. The first aim of this proposal is to characterize three new human proteins. These proteins form a tightly bound complex that is associated with the U4/U6 snRNP. Two of the proteins are homologs of known yeast splicing factors; the third protein is a new human cyclophilin. Theses are the first U4/U6-specific proteins found in humans, and study of them will provide important insights into the mechanism of splicing. Cyclophilins are thought to mediate interaction among proteins. The U4/U6 snRNP undergoes a number of large conformation changes in the splicing pathway, and this new cyclophilin could facilitate these conformational changes. Cyclophilins have been studied extensively, because they are the target of the immunosuppressive drug cyclosporin A. However, their cellular functions are generally obscure. Study of this cyclophilin will provide valuable insights into the normal cellular roles of the cyclophilins. The second aim of this proposal is to study the second step of pre-mRNA splicing. The Prp18 protein, previously shown to be required for the second catalytic reaction in splicing, will be the center of this study. Homologs of Prp18 have been characterized in both humans and yeast. The interactions of Prp18 with other components of the splicing machinery will be studied; RNA interactions will be probed with UV-crosslinking, while interactions with other proteins will be studied with the two hybrid system. Prp18 will also be used as a tool to study conformation of the spliceosome at the time of the second reaction. A new affinity method for purifying spliceosomes at the time of the second reaction will allow probing of the interactions in the spliceosome at the time of a catalytic step. Following these interactions through the second step of the reaction will further the understanding of the mechanism of splicing.